WO2011071134A1

WO2011071134A1 - Prophylactic composition for influenza infection

Info

Publication number: WO2011071134A1
Application number: PCT/JP2010/072192
Authority: WO
Inventors: 正雄高見; 真也永渕; 諭高杉; 裕康赤津
Original assignee: 明治乳業株式会社
Priority date: 2009-12-10
Filing date: 2010-12-10
Publication date: 2011-06-16
Also published as: CN102665738A; JP5951087B2; JP2016028033A; TWI476000B; JPWO2011071134A1; CN102665738B; HK1171961A1; TW201127389A

Abstract

It was clarified that a culture of a propionic acid bacterium exhibited a prophylactic effect on influenza infection. Disclosed is a composition comprising a culture of a propionic acid bacterium which is capable of potentiating the induction of a neutralizing antibody in an animal that has been inoculated with an influenza vaccine. The fact that the component constituting the aforesaid composition has been eaten by humans over a long time assures the high safety and good taste thereof and, therefore, said composition can be taken over a long period of time. In addition, the aforesaid composition can contain, as optional components, a milk fermentation product and an oligosaccharide. It can be also expected that another effect of controlling the functions of the intestines is achieved by administering the aforesaid composition.

Description

Influenza preventive composition

The present invention relates to a composition containing a culture of propionic acid bacteria. Alternatively, the present invention relates to a composition for preventing influenza infection or a method for preventing it.

Influenza is a highly infectious disease that uses influenza virus as a pathogen. Influenza virus is a minus-strand single-stranded RNA virus belonging to the Orthomyxoviridae family. Orthomyxoviridae consists of 1 genus of influenza virus (1 genus and 1 family). Depending on the antigenicity of the nucleoprotein, there are three subtypes of A-C in influenza viruses. In contrast to types B and C, which exclusively infect humans as hosts, type A influenza viruses can infect a wide range of organisms such as mammals and birds. For this reason, gene hybridization occurs in birds and mammals, and mutant viruses with different pathogenicity and antigenicity are likely to be generated.

For example, swine influenza, which has been prevalent worldwide in recent years, is an influenza that develops due to infection with a type A H1N1 subtype influenza virus. The swine influenza virus is thought to have infected humans and led to a pandemic. In addition, avian highly toxic influenza that is predicted to have a pandemic is thought to be a potentially pandemic if avian highly toxic influenza viruses gain infectivity to humans (Takaki Sugawara et al., WHO).

Currently, therapeutic drugs that inhibit the growth of influenza virus are commercially available. It is said that a currently available therapeutic drug for influenza can be expected to have a sufficient therapeutic effect when appropriate medication is performed at an early stage after infection. However, influenza remains a dangerous infection, especially for high-risk groups such as the elderly, children, and people with chronic illnesses. The emergence of viruses that are resistant to therapeutic drugs has also been reported.

Even now that therapeutic drugs have been developed, it is said that inoculation with influenza vaccine is meaningful in order to prevent the severity of influenza. Inoculation with influenza vaccine induces neutralizing antibodies against influenza virus and can prevent the virus from spreading and spreading after infection (C, Avendano et al., Zhu Q, et al.). For viruses that are similar in antigenicity to the virus inoculated as a vaccine, it is said that the effect of preventing seriousness is high. There is a report that the induction rate of neutralizing antibodies in healthy adults by vaccination (70-90%) is lower (17-53%) in the elderly (Goodwin K, et al.). It is particularly important to raise the neutralizing antibody induction rate of the elderly because the immunity of the elderly is reduced and infection can become severe and lead to death.

WO 2001/028547 WO 2004/085364 WO 2007/023935 WO 2005/094850

An object of the present invention is to provide a composition containing a culture of propionic acid bacteria. In particular, it is an object of the present invention to provide a composition having an effect of preventing influenza virus infection. Alternatively, an object of the present invention is to provide a composition having an intestinal regulating action.

The virus infection prevention effect of influenza vaccines mainly depends on the induction of virus neutralizing antibodies. Therefore, stimulating the induction of neutralizing antibodies after vaccination is considered effective for enhancing the preventive action of the vaccine. Alternatively, a virus neutralizing antibody that is induced as a normal immune response in a patient's body at the time of viral infection is one of the important biological defense functions of the patient. The present inventors have conducted research on components that can enhance the induction of virus neutralizing antibodies by influenza vaccine and virus infection. As a result, the inventors have found that the induction of virus neutralizing antibodies upon inoculation with influenza vaccine is enhanced in animals administered with a culture of a specific microorganism, thereby completing the present invention. That is, the present invention relates to the following compositions and uses thereof.

[1] A composition for preventing influenza infection comprising a culture of propionic acid bacteria.
[2] The composition according to [1], wherein the propionic acid bacterium is Propionibacterium freudenreichii.
[3] The composition according to [1], which additionally contains a milk fermentation component and / or an oligosaccharide.
[4] The composition according to [3], wherein the milk fermentation component is milk fermented with lactic acid bacteria belonging to the genus Lactobacillus and / or lactic acid bacteria belonging to the genus Streptococcus, or a mixture thereof.
[5] The composition according to any one of [3] to [4], wherein the milk fermentation component is non-aged cheese.
[6] The composition according to [3], wherein at least one of the sugars constituting the oligosaccharide is galactose.
[7] The composition according to any one of [3] to [4], wherein the composition contains a culture of propionic acid bacteria and a milk fermentation component, and both are sterilized.
[8] The composition according to any one of [1] to [5], which is used for enteral administration to an animal vaccinated with an influenza vaccine.
[9] The composition according to [3], comprising the following nutrients;
A culture of propionic acid bacteria;
Milk fermentation ingredients;
oligosaccharide;
protein;
Carbohydrates;
Lipids; and dietary fiber.
[10] The composition according to [9], further comprising at least one nutrient selected from the group consisting of vitamins, minerals, organic acids, and organic bases.
[11] An agent for promoting the induction of an influenza virus neutralizing antibody in an animal vaccinated with an influenza vaccine, comprising a culture of propionic acid bacteria.
[12] A method for preventing influenza infection including the following steps;
(1) administering a propionic acid bacteria culture to an animal; and
(2) A step of inoculating an animal with an influenza vaccine.
[13] The method for preventing influenza infection according to [12], wherein at least one step (1) is performed before, after, or simultaneously with step (2).
[14] Prevention of influenza infection according to [12] or [13], wherein in the step (1), a culture of propionic acid bacteria is administered together with one or both of a milk fermentation component and an oligosaccharide Method.
[15] A composition comprising the following components (a)-(c):
(a) an oligosaccharide,
(b) milk fermentation ingredients, and
(c) Propionic acid bacteria culture.
[16] The composition according to [15], wherein at least one of the sugars constituting the oligosaccharide is galactose.
[17] The composition according to [15] or [16], which is a composition for intestinal regulation.

The composition of the present invention enhances the neutralizing antibody inducing action of the influenza vaccine. The composition of the present invention can be produced by blending ingredients that have already been administered to humans as a liquid food or the like. Therefore, the composition of the present invention is already guaranteed a high level of safety. Therefore, the composition of the present invention can be administered not only simultaneously with vaccination but also continuously before and after the vaccination. By continuous administration, the effect of promoting neutralization antibody induction by the composition of the present invention can be expected continuously.

In order to enhance the immune response to viral vaccines including influenza viruses, vaccines are often combined with an adjuvant. Needless to say, a high degree of safety is required for an adjuvant to be added to a vaccine. Therefore, careful safety testing is required for the development of new adjuvants. On the other hand, since each component constituting the composition of the present invention has already been eaten as a food, it has already been proved that there is no risk even if it is continuously administered enterally to the human body. Has been. Further, the composition of the present invention can be administered enterally in combination with a known adjuvant. In other words, the present invention is significant not only in terms of safety but also in that it provides a new option as a means for enhancing an immune response to a vaccine.

In the preferred embodiment, the composition of the present invention is provided by a combination of components that have already been widely consumed as liquid food or food. Therefore, by continuously ingesting the composition of the present invention as a liquid food, it is possible to create a state in which the ability to induce neutralizing antibodies against influenza virus is constantly enhanced. Infection with influenza viruses is a serious problem in a population of people at risk of getting severe influenza, such as older people and hospitalized patients. However, if the composition of the present invention is prophylactically administered as a liquid food, the ability of the entire population to prevent infection can be enhanced.

It is a figure which shows a test protocol. (The administration period of each liquid food, the time of vaccination and antibody measurement, and the time of intestinal flora analysis.) It is a graph which shows the influence which administration of a milk fermentation component and a propionic acid bacteria culture | cultivation has on the neutralizing antibody titer after vaccination. In the figure, the vertical axis represents the logarithmic value of antibody titer (log10), and the horizontal axis represents the elapsed time (week) after vaccination. The statistically significant difference between the control group and the test group is Mann-Whitney U test, and the statistically significant difference between the 2nd and 6th week after vaccination is the Wilcoxon signed rank sum test. Analyzed. * Indicates that there was a significant difference (p <0.05) in the neutralizing antibody titer at 6 weeks compared to 2 weeks. A: neutralizing antibody titer against H1N1, B: neutralizing antibody titer against H3N2, C: neutralizing antibody titer against B1 antigen It is a graph which shows the change of the stool score during a test period. In the figure, the vertical axis represents the Bristol stool scale (1-7), and the horizontal axis represents the administration period (weeks) of each liquid food. The statistically significant difference between the control group and the test group was analyzed by Mann-Whitney U test, and the statistically significant difference of each week before and during the test was analyzed by Wilcoxon signed rank sum test. Furthermore, * indicates a significant difference (p <0.05) between the control group and the test group, and a indicates that there was a significant difference (p <0.05) with respect to the time of grouping (−4 weeks). It is a graph which shows the influence which a milk fermentation component and a propionic acid bacteria culture give to the blood cytokine density | concentration at the time of influenza vaccination. In the figure, the vertical axis indicates A: IL-7 concentration (pg / mL), B: IL-17 concentration (pg / mL), and C: TGF-β1 concentration (ng / mL). The horizontal axis shows the elapsed time (weeks) at the time of blood collection with the grouping time set to -4. The statistically significant difference between the control group and the test group is the Student's test (equal variance) or the Welch test (non-equal variance), and the statistical significance between each week before and during the study. Differences were analyzed by paired t test.

The present invention provides a composition for preventing influenza infection or a preventive agent for influenza infection, comprising a culture of propionic acid bacteria.
The preventive agent or composition of the present invention includes a culture of propionic acid bacteria. Propionic acid bacteria are gram-positive anaerobic bacteria belonging to the genus Propionibacterium, which are microorganisms that produce propionic acid oxygen-freely from sugars. Specifically, the following microorganism cultures can be added to the composition of the present invention.
Propionibacterium freudenreichii,
Propionibacterium toenii (P. thoenii),
Propionibacterium acidipropionici (P. acidipropionici),
Propionibacterium jensenii, etc. These propionic acid bacteria are microorganisms used in cheese production. In addition, the following microorganisms can also be shown as propionic acid bacteria.
Propionibacterium avidum (P. avidum),
Propionibacterium acnes (P. acnes),
Propionibacterium lymphophilum (P. lymphophilum),
Propionibacterium granulosam (P. granulosam)

A method for isolating these microorganisms from nature and fermented milk is known. Propionic acid bacteria can also use microorganisms that are used in Swiss cheese production and the like. The culture of propionic acid bacteria in the present invention refers to those obtained by culturing the above propionic acid bacteria under appropriate culture conditions. Methods for culturing propionic acid bacteria are known. In the culture of propionic acid bacteria, the conditions described in WO03 / 016544A1 and the like can be applied. For example, as a medium for culturing propionic acid bacteria, a composition in which beer yeast extract or the like is added to skim milk powder or a proteolytic processed product of skim milk powder is known. A culture of propionic acid bacteria can be obtained by inoculating a suitable medium with Propionibacterium re freudenreichii and culturing them under conditions that allow propionic acid bacteria to grow.

Alternatively, as a method of culturing propionic acid bacteria at a high concentration, there is a method of culturing propionic acid bacteria in a medium in which whey protein concentrate (Whey Protein Concentrate: PCWPC) or its enzyme degradation product is added with minerals and monosaccharides. (Japanese Published Patent No. 10-304871). For example, a culture obtained by culturing Propionibacterium freudenreichii in a medium containing a whey protein concentrate is preferable as the culture of propionic acid bacteria in the present invention. Alternatively, as an efficient method for culturing propionic acid bacteria, there is a method in which bifidobacteria and propionic acid bacteria are cultured while circulating the culture solution in different culture tanks (Japanese Patent Publication No. 8-66178). A culture of propionic acid bacteria obtained by these culturing methods can also be added to the composition of the present invention.

For example, propionic acid bacteria can be cultured at high density by adding a processed product of whey as the main component of the medium. Examples of processed products of whey include the following components.
Whey powder,
Protease-treated product of whey and whey powder In addition to whey protein, a mixture of minerals and monosaccharides can be added to the medium. In order to reduce the sugar concentration in the medium, a whey protein concentrate (hereinafter sometimes referred to as WPC) can also be added as a whey protein source. WPC can be obtained by dialysis of whey to reduce the lactose content. The whey protein concentrate can be further separated into protein components with high purity to obtain a whey protein isolate (hereinafter sometimes referred to as WPI). These components are added to the medium, and an appropriate amount of saccharide and a deficient mineral are individually added to the medium, and the composition of the medium can be used for the culture of propionic acid bacteria.

Whey is a water-soluble component that remains when, for example, fat, casein, fat-soluble vitamins, and the like are removed from milk. Whey generally produced acid casein and quark from cheese whey, rennet whey (or sweet whey) and skim milk obtained as a by-product when natural cheese and rennet casein were produced. Casein whey and quark whey (or acid (acid) whey) obtained in some cases. The main components of whey are protein (β-lactoglobulin, α-lactalbumin, etc.), lactose, water-soluble vitamins, and salts (mineral components), each characteristic of which is a component of milk rather than research as a component of whey As revealed in research.

"Whey-related products" include whey concentrated whey, whey powder dried whey, and whey powders that have been concentrated by ultrafiltration (UF), etc. Protein concentrate (Whey Protein ： Concentrate: hereinafter also referred to as “WPC”), whey is removed by fat removal by microfiltration (MF) or centrifugation, concentrated by UF and dried WPC (low fat, high protein), whey protein isolate (WPI Protein Isolate), which was subjected to selective fractionation by ion-exchange resin method or gel filtration method, followed by drying treatment. ), Desalted whey that has been desalted by nanofiltration (NF) method or electrodialysis method, etc. And mineral concentrated whey that has been concentrated by centrifugation. Of these, WPC containing 15% to 80% of milk protein in dry weight (solid content) is a protein-enriched whey powder on March 30, 1998, due to a partial revision of the Ministry of Milk Ordinance. Defined in the product (concentrated whey, whey powder, WPC, whey protein concentrated powder, regardless of the presence or absence of a desalting process, as long as they have undergone the manufacturing process specified in the Ministerial Ordinance such as milk).

The whey protein concentrate (WPC) is obtained by concentrating main protein of whey and the like by an ultrafiltration (UF) method and then drying. Generally, it is a collective term for those in which about 25% or more of the solid content is whey protein. It can be obtained by reducing lactose and salts from whey and relatively strengthening whey protein so that the solid content is about 25% to about 80%. In particular, WPC containing milk protein at a dry weight of 15% to 80% is defined as a protein-enriched whey powder according to a ministerial ordinance such as milk.
The standard method for producing whey protein concentrate (WPC) is as follows.
(1) A step of concentrating whey after membrane separation. Or (2) A step of concentrating and drying the whey after membrane separation.
In addition, a general apparatus and method can be used for the concentration treatment, for example, a vacuum evaporator (evaporator), a vacuum kettle, a thin film vertical ascending tubular concentrator, a thin film vertical descending tubular concentrator, a plate concentrator. The method of heating under reduced pressure can be used. Also, a general apparatus and method can be used for the drying process, for example, spray drying (spray dryer) method, drum drying method, freeze vacuum drying (freeze dryer) method, vacuum (reduced pressure) drying method, or the like. be able to.

A whey protein isolate (WPI) is obtained by concentrating main protein of whey and the like by an ion exchange resin method, an electrodialysis method, and the like, and then drying. Generally, it is a general term for what is about 85% to about 95% of solids is whey protein. It can be obtained by reducing lactose and salts from whey and relatively strengthening whey protein to about 90% solids (85% to 95%).
The standard method for producing whey protein isolate (WPI) is as follows.
(1) A step of concentrating whey after membrane separation, ion exchange resin treatment or electrodialysis treatment. Or (2) A step of concentrating and drying whey after membrane separation, ion exchange resin treatment or electrodialysis treatment.
In addition, a general apparatus and method can be used for the concentration treatment, for example, a vacuum evaporator (evaporator), a vacuum kettle, a thin film vertical ascending tubular concentrator, a thin film vertical descending tubular concentrator, a plate concentrator. The method of heating under reduced pressure can be used. Also, a general apparatus and method can be used for the drying process, for example, spray drying (spray dryer) method, drum drying method, freeze vacuum drying (freeze dryer) method, vacuum (reduced pressure) drying method, or the like. be able to.

In the present invention, the following composition can be shown as a medium composition suitable for culturing propionic acid bacteria. All the numerical values shown below are weight ratios (W / W%). Hereinafter, when the composition is expressed as a percentage, it is a weight ratio (W / W%) unless otherwise specified.
Protein content: 1-5%, preferably 1.5-4.0%
Carbohydrate content: 1 to 4%, preferably 1.5 to 3.0%
The addition amount of whey powder, whey protein, or their protease-treated product is adjusted so that such a content can be obtained. Moreover, as a saccharide | sugar, instead of lactose, glucose or the monosaccharide which lactose-treated lactose is preferable.

In order to reduce the cost of the medium and to obtain a culture suitable for food, a lactase treatment solution of whey mineral can be used as a source of carbohydrates and minerals. Specifically, WPC can be used as a protein source, and whey minerals can be used as a sugar source and a mineral source. If the mixture of the two optimization rates is used as a medium raw material, propionic acid bacteria can be cultured at a higher concentration than when whey powder is used as the medium raw material. A detailed medium preparation method for culturing propionic acid bacteria is shown below.

WPC (bovine) degrades protein with protease after reduction. Protease is an endo & exo type derived from Aspergillus oryzae and the amount used is 3% of the amount of protein to be degraded. The reaction is carried out at 50 ° C. and pH 7.0, and stirring is continued for 3 to 5 hours until no decrease in pH is observed. Whey minerals break down lactose with lactase. The amount of lactase used is 2 to 8% of the sugar mass to be decomposed, the reaction is carried out at 50 to 60 ° C. (preferably 55 ° C.), pH 5 to 6, and stirring is continued until the protein is completely decomposed.

Subsequently, preferably, the final medium concentration is a protein concentration of 1 to 5% (preferably 1.5 to 4.0%) and a carbohydrate concentration of 1 to 4% (preferably 1.5 to 4.0%). These two liquids are mixed so that it may become 3.0%. Finally, components commonly used for culturing propionic acid bacteria such as yeast extract, sodium sulfate, and asparagine are added to the medium, and the pH is adjusted to 5 to 8 (preferably 5.5 to 7.5). The preparation of is finished. The culture process of propionic acid bacteria is as follows.

That is, the medium temperature is set to 20 to 40 ° C., and the starter is inoculated so that the viable cell count immediately after the start of culture is 10 ⁷ to 10 ⁸ cfu / ml, and cultured for 3 to 4 days. The pH is maintained at 5.5 to 7.5 with an aqueous potassium carbonate solution. Additional glucose can be added during the culture. Propionic acid bacteria in the culture obtained in this way reach about 5 times the conventional amount.

The culture conditions as described above are particularly suitable for culturing propionic acid bacteria for cheese. As propionic acid bacteria for cheese, in addition to Propionibacterium freudenreichii, Propionibacterium acidipropionici, Propionibacterium jensenii, Propionibacterium thoenii, and the like can be used. More specifically, a culture capable of obtaining the following strains as propionic acid bacteria can be used in the present invention.
Propionibacterium freudenreichii ATCC 6207
P. freudenreichii ATCC 8262
P. freudenreichii IFO 12424
P. freudenreichii IFO 12426
P. freudenreichii IFO 12391
P. freudenreichii ET-3 (FERM BP-8115)
These propionic acid bacteria can be cultivated alone, or a plurality of strains can be mixed and cultured. Alternatively, the obtained culture can be mixed after culturing a plurality of microorganisms alone. The culture thus obtained can be directly used for eating and drinking as it is. This can be further pulverized or liquefied and processed into a functional raw material that is easy to handle. That is, the culture obtained by culturing the propionic acid bacterium can be blended in the composition of the present invention as it is or after processing.

Furthermore, those skilled in the art can appropriately adjust the medium composition and culture conditions in order to further optimize these known methods. For example, with respect to the nitrogen source, various amino acids and salts thereof can be added in addition to casein protein, WPC, etc., and the propionic acid bacteria proliferating ability and the effect of preventing influenza infection can be enhanced. Not only the medium composition but also the culture conditions can be adjusted and optimized. The culture conditions include the oxygen concentration, temperature, pressure, etc. of the culture atmosphere.

As long as the propionic acid bacteria culture of the present invention maintains the effect of preventing influenza virus infection, a fraction thereof can also be used. Therefore, the culture of propionic acid bacteria includes, for example, the culture itself of propionic acid bacteria, the culture supernatant, the cells, the extract thereof, the dry powder thereof, or the dilution thereof. Here, the “propionic acid bacteria culture itself” means a mixture of propionic acid bacteria and medium components. The dispersion state of propionic acid bacteria in the medium component is arbitrary. That is, the propionic acid bacteria may be dispersed or precipitated in the medium components. On the other hand, “culture supernatant” usually refers to a state in which cells of propionic acid bacteria are removed from “culture of propionic acid bacteria itself” by filtration or centrifugation. Furthermore, “bacteria” means propionic acid bacteria isolated from “the culture of propionic acid bacteria itself”. When separating the medium components and the cells of propionic acid bacteria, the separation of the two is usually allowed to be incomplete. Therefore, for example, it is allowed that the medium components are mixed into the cells of propionic acid bacteria isolated from the culture.
For example, the effect of promoting the induction of neutralizing antibodies after influenza vaccination of a fraction of a culture of propionic acid bacteria is 30% or more, for example, compared to the culture of the same propionic acid bacteria (before fractionation), When it is preferably 50% or more, more preferably 70% or more, it can be said that the preventive effect of the influenza virus infection of the culture was maintained.

The cultured culture of propionic acid bacteria after culturing can be sterilized and added to the composition of the present invention. Alternatively, the milk-fermented component and the composition after blending can be sterilized. For example, in the case of milk, it is stipulated in a ministerial ordinance such as milk, and the following heat sterilization treatment is generally performed.
Low temperature sterilization,
High temperature sterilization,
High temperature short time sterilization,
Ultra high temperature instant sterilization

These sterilization methods or sterilization methods can be applied to the culture of propionic acid bacteria in the present invention or a composition containing the same. The sterilization treatment can be performed in batch units or continuously. The treatment temperature and treatment time vary depending on the sterilization method, but are preferably selected in the range of 50 ° C. to 200 ° C. and 0.1 second to 1 hour according to the sterilization method. When sterilizing a culture of propionic acid bacteria, it is desirable to maintain a state in which the amount of dissolved oxygen in the liquid is reduced. Therefore, it is preferable to maintain the inert gas atmosphere continuously during the heat sterilization. Examples of the inert gas include nitrogen gas, argon gas, carbon dioxide gas, and the like. Among them, nitrogen gas is a preferable inert gas because it is present in a large amount in the air, is relatively low in cost, has been confirmed to be safe, and does not affect the flavor and quality of food and drink. . The present inventors have confirmed that the preventive effect of influenza is maintained in a culture of propionic acid bacteria after sterilization.

In the present invention, the preventive action against influenza infection of the culture of propionic acid bacteria is maintained even after sterilization. Usually, the effect of lactic acid bacteria on the host depends on the action of live bacteria. Therefore, the discovery of useful functions in the sterilized culture was an unexpected finding. It has been reported that probiotics and prebiotics improve the intestinal environment and activate the immune system. In general, probiotics refer to microorganisms that have a beneficial effect on the host by being introduced into the intestine of the host in a living state. In contrast to probiotics, prebiotics refer to substances that have beneficial effects on the host by acting on microorganisms that originally lived in the intestines. For example, several lactic acid strains have been reported as probiotics having an effect of promoting neutralizing antibody induction against influenza vaccines, particularly in a test using humans. However, since these lactic acid bacteria are probiotics that act as live bacteria, production and quality control are not easy. In addition, live bacterial preparations generally have limited shelf life. For example, even if a viable preparation is stored at a low temperature after production, it cannot often withstand long-term storage.

The culture of propionic acid bacteria in the present invention with respect to lactic acid bacteria is prebiotic. In sterilized cultures (prebiotics), there is no change in quality due to microbial activity. Therefore, in prebiotics, the preventive effect of influenza infection can be stably maintained. That is, the composition of the present invention is easy to manufacture and quality control. Furthermore, the composition of the present invention, which is a prebiotic, can be stored at room temperature for a long time. In general, when an effect of viable lactic acid bacteria is expected by oral administration, the influence of gastric acid must be considered. This is because lactic acid bacteria are reduced by gastric acid and a sufficient amount of viable bacteria cannot be delivered into the intestine. On the other hand, in the present invention, the preventive effect of propionic acid bacteria on influenza infection does not depend on the action of live bacteria. Therefore, sufficient influenza preventive effect can be achieved even by oral administration.

The culture of propionic acid bacteria can be processed into powder or liquid after sterilization. For example, an appropriate excipient may be added to the culture solution to adjust the culture solid content to 30 to 40% by weight and then dried to form a powder. As the excipient, skim milk powder, whey powder, raw starch, dextrin and the like can be used. As the excipient, in addition to the above, WPC, whey protein isolate (Whey Isolate: WPI), and modified starch can be used as necessary. Methods for drying cultures are also known. For example, the culture solution can be spray-dried as it is. Specifically, for example, soluble starch, British gum, oxidized starch, starch ester, starch ether and the like can be used as the modified starch. Alternatively, the culture solution and the reducing solution of the excipient are mixed, concentrated until the solid content becomes 30 to 40% by weight, and then spray-dried. The dried culture can be stably stored for a long period of time by deoxidation treatment (eg, nitrogen sealing or addition of a deoxidizing agent) at the time of filling. Moreover, it can also be processed into a triturated preparation (0.2% powdered powder) for easy use in foods.

It is known that a culture of propionic acid bacteria has an effect of promoting the growth of lactic acid bacteria. A culture of a propionic acid bacterium having such an action or a processed product thereof is called a “Bifidogenic Growth Stimulator (BGS)”. BGS can be used as a culture of propionic acid bacteria in the present invention as long as it has an action of promoting production of neutralizing antibodies against influenza virus in animals vaccinated with influenza vaccine.

As the culture of propionic acid bacteria in the present invention, a whey fermented product of propionic acid bacteria is preferable. For example, a propionic acid bacterium culture obtained by fermenting a Propionibacterium freudenreichii -3 ET-3 strain producing a Bifidogenic Growth: Stimulator (BGS) with a 10% whey powder reducing solution is used as the composition of the present invention. Can be contained as an active ingredient. For example, a culture of propionic acid bacteria containing BGS is called “profec” and is permitted as an ingredient involved in food for specified health use. BGSpowder (made by Meiji Dairies, trade name) and tummy vitality tablets (made by Meiji Dairies, trade name; “Tanaka vitality” is a registered trademark of Meiji Dairies Co., Ltd.) are commercially available as compositions containing “Profec”. . Therefore, “B.G.S.powder” or “tummy vitality tablet” can also be used as the composition of the present invention.

Especially, BGS contained in Profec is 1,4-dihydroxy-2-naphthoic acid; 1,4-dihydroxy-2-naphthoic acid (DHNA) and 2-amino-3-carboxy-1,4-naphthoquinone 2-amino-3-carboxy-1,4-naphthoquinone (ACNQ). Among these, DHNA is a biosynthetic intermediate of vitamin K2 (menaquinone) in microorganisms. These substances promote proliferation by efficiently reoxidizing NADH produced in the energy metabolism process of bifidobacteria. Therefore, either or both of the following components (i) and (ii) can be used as the propionic acid bacteria culture. That is, the present invention
Prevention of influenza infections including (i) 1,4-dihydroxy-2-naphthoic acid (DHNA) and (ii) 2-amino-3-carboxy-1,4-naphthoquinone (ACNQ) or both Provide the agent.

In the present invention, the dose of the prophylactic agent for influenza infection containing the above components (i) and / or (ii) is usually used for adults with the amount of DHNA contained in the propionic acid bacteria culture as an index. In this case, the amount of DHNA contained in the propionic acid bacteria culture is generally in the range of 0.01 μg / kg-100 mg / kg. In some cases, this may be sufficient, or vice versa. It can also be administered in 2-4 divided doses per day. The dose can be set in consideration of the patient's condition such as age and weight, administration route, expected degree of preventive effect, and the like.

Profec is specifically approved as an ingredient in foods for specified health use because it specifically increases Bifidobacterium in the human intestine (Nobuo Yoda: ILSI, No. 80, 5- 13 (2004)). Currently, “BGSpowder” and “tummy vitality tablet” are commercially available as compositions containing Profec / Profec. Therefore, there is no difficulty in obtaining. However, it is not known that neutralizing antibody induction is enhanced by feeding Profec to animals vaccinated with influenza. That is, the present invention
Inoculate an influenza vaccine containing either (i) 1,4-dihydroxy-2-naphthoic acid (DHNA) and (ii) 2-amino-3-carboxy-1,4-naphthoquinone (ACNQ) or both A neutralizing antibody induction promoter in a treated animal is provided. Alternatively, the present invention relates to a pharmaceutical composition for promoting the induction of neutralizing antibodies in an animal vaccinated with influenza vaccine, comprising either or both of the above components (i) and (ii).

When 1,4-dihydroxy-2-naphthoic acid (DHNA) is added to the composition of the present invention, it is desirable to keep the dissolved oxygen level in the composition low. This is because DHNA is decomposed by dissolved oxygen and its concentration decreases during storage. Methods for reducing the dissolved oxygen level in compositions containing DHNA are known (WO2004 / 85364). Specifically, when the composition is liquid, the dissolved oxygen can be replaced with a gas other than oxygen by bubbling the liquid composition with a gas not containing oxygen. Nitrogen is preferred as the gas not containing oxygen. Furthermore, the dissolved oxygen level can be kept low by adding an antioxidant compound together with DHNA. A known antioxidant can be used for the compound having antioxidant properties. Specifically, hyposulfite, ascorbic acid (vitamin C), erythorbic acid, carotene, tocopherol, and polyphenols having an antioxidant action are known as antioxidants.

As polyphenols, in addition to synthetic products, polyphenols derived from natural products can also be used. For example, polyphenols derived from teas, grapes, lemons, coffee, Murasakimochi, soybeans and the like are known. Juices such as fruits, vegetables, seeds, plant leaves and the like containing a large amount of these polyphenols or extracts thereof can be blended as polyphenols in the composition of the present invention. For example, a polyphenol extract can be obtained by extraction with water or an organic solvent. Furthermore, concentrates, purified products, and dried products of these natural polyphenol-containing products can be used as polyphenols.

The amount of the antioxidant added can be reduced by adding the same amount or more than the amount used for the usual antioxidant application, depending on the type of the antioxidant. For example, when ascorbic acid is added alone to the composition without bubbling an inert gas and DHNA stability is expected, 0.01% by weight or more of ascorbic acid is added to the total weight of the solution. Can be added. Antioxidants can be added to the composition prior to adding DHNA to the composition. Alternatively, it can be added to the composition together with DHNA to prevent its degradation. For example, when ascorbic acid is added as an antioxidant, the amount added may be about 100 kcal (or per 100 g of composition), for example, 1 μg-2 g, usually 150 μg-1.5 g, preferably about 1 mg-500 mg. .

The culture of propionic acid bacteria can be blended in a proportion of, for example, 0.001 to 20%, usually 0.01 to 15%, preferably 0.01 to 10%, based on the entire composition. Alternatively, the milk fermentation component contained in the entire composition of the present invention is such that when quark is added as the milk fermentation component, the concentration of the protein portion of the quark is about 0.01% to about 30% with respect to the whole composition. , Preferably about 0.1% to about 20%, more preferably about 0.5% to about 10%.

The composition of the present invention can be in any dosage form such as liquid, paste, or dried solid. The composition of the present invention can be formulated into a culture of propionic acid bacteria by blending with a carrier suitable for enteral administration or a pharmaceutically acceptable carrier. More specifically, it can be formulated into tablets, capsules, granules, powders, syrups and the like. Or it can also supply in the state which disperse | distributed the culture of propionic acid bacteria to the milk fermentation component. These various preparations are pharmaceutical preparations such as excipients, binders, disintegrants, lubricants, flavoring agents, solubilizers, suspension agents, coating agents, solvents, isotonic agents, etc. It can be formulated using known adjuvants that can be commonly used in the technical field. It can also contain an appropriate amount of minerals such as calcium. Furthermore, an appropriate amount of vitamins, minerals, organic acids, sugars, amino acids, peptides and the like can be added. Organic acids include fatty acids such as short chain fatty acids.
It is known that a fermented whey product of propionic acid bacteria can be expected to have an excellent intestinal action. However, it is not known that a culture of propionic acid bacteria exhibits a preventive action against influenza infection.

In addition, a milk fermentation component and an oligosaccharide can be added to the composition of the present invention. That is, the present invention relates to a composition comprising the following components (a) to (c).
(a) an oligosaccharide,
(b) milk fermentation ingredients, and
(c) Propionic Acid Bacteria Culture Generally, oligosaccharide is also called oligosaccharide and refers to a compound in which 2 to 20 sugars are glycosidically bonded. For example, in the present invention, the following saccharides can be used as oligosaccharides.
Dairy oligosaccharides,
Isomaltoligosaccharide,
Fructo-oligosaccharide,
Galactooligosaccharides,
Xylooligosaccharides,
Soybean oligosaccharides,
Nigero-oligosaccharide,
Gentiooligosaccharides,
Lactose,
sucrose,
Maltose, etc.

In oligosaccharides, there are compounds that are easily decomposed under acidic conditions. Therefore, when the composition of the present invention or a food containing the same is acidic, it is preferable to use an oligosaccharide that is stable under acidic conditions. For example, an oligosaccharide containing galactose as a constituent sugar is preferable as an oligosaccharide compounded under acidic conditions. In the present invention, the oligosaccharide containing galactose as a constituent sugar includes a compound in which 2 to 20 sugars are glycoside-bonded and includes one or a plurality of galactose in the sugar constituting the compound. More specifically, raffinose family oligosaccharide (soybean oligosaccharide), galactooligosaccharide and the like can be shown as preferred oligosaccharides. Of these, galactooligosaccharide is a preferred oligosaccharide in the present invention.

In the present invention, galacto-oligosaccharide refers to an oligosaccharide having galactose as a main constituent sugar. Lactose (Gal (β1-4) Glc) is the basic structure, and oligosaccharides with a structure in which one to several galactose residues are bonded to this, or disaccharides with β-1,3 bonds between galactose and glucose ( Transfer saccharides), Gal- (Gal) n-Glc (n = 1-18), Gal- (Gal) n-Gal (n = 1-18) can also be included in the examples of galactooligosaccharides. In the present invention, the number of galactose residues constituting the galactooligosaccharide is usually 1-20, preferably 1-10, more preferably 1-8. Moreover, the ratio of the galactose which comprises a galactooligosaccharide can show 10% or more with respect to the total of the monosaccharide which comprises a galactooligosaccharide, for example, Preferably it is 30% or more, More preferably, it can show 50% or more. An example of a method for producing galactooligosaccharides is by reacting lactose with β-galactosidase, which has a high ability to transfer galactose (Sayairi Hayato, functions of galactooligosaccharides and application to food, FOOD STYLE 21, 2, pp .76-78 (1998)). As β-galactosidase, for example, those derived from microorganisms such as Cryptococcus laurentii, Bacillus circulans and the like are used.

In addition, in the standard of the food for specified health use (standard standard type), the oligosaccharide produced from the action of β-galactosidase (β-D-galactoside galactohydrolase, EC3.2.1.23, derived from cryptococcus yeast) from lactose is galacto-oligo. Defined as sugar. The galactooligosaccharide thus produced is an oligosaccharide in which one or more galactoses are glycosidically linked to the galactose residue of lactose, and 4′-galactosyl lactose (Gal (β1-4) Gal (β1-4) Glc) It is said to be the main ingredient (Notice of food safety from 0701007 dated July 1, 2005, Ministry of Health, Labor and Welfare, Ministry of Health, Labor and Welfare, Director of Food Safety Department, “Standard for the establishment of food for specified health use (standard type)” About setting standards ”).

Galactooligosaccharide is contained in breast milk, has an effect of appropriately increasing the number of bifidobacteria in the intestine, and is known as a sugar that is difficult to digest and absorb. It is also known that galactooligosaccharide is useful for mineral supplementation after gastrectomy (International Publication WO 98/15196). Furthermore, fructooligosaccharides are hydrolyzed under strong acidity (Atsuko Nakamura, Changes in sugars in sour pork and boiled foods using fructooligosaccharides, Bulletin of Tokyo Kasei Gakuin University, 43, pp. 49-53 (2003 )), Galactooligosaccharides have the property that they are less susceptible to degradation due to degradation under acidic conditions or heating conditions (Hayato Sawairi, functions of galactooligosaccharides and application to food, FOOD STYLE 21, 2, pp.76-78) (1998)). By adding an oligosaccharide to the composition of the present invention, the intestinal regulating action of the composition can be further enhanced. That is, the composition of the present invention can be used as a composition for intestinal regulation. Gastrointestinal symptoms such as diarrhea are one of the typical infectious symptoms of influenza virus. Therefore, it can be said that a composition having an intestinal regulating action is a desirable feature as a composition for preventing influenza infection.
The concentration of the oligosaccharide contained in the composition of the present invention is about 0.001 to about 20.0%, preferably about 0.05 to about 11%, more preferably about 0.1 to About 6%. The blending amount of the oligosaccharide can be adjusted according to the dosage form, symptoms, body weight and the like.

In addition to the culture of propionic acid bacteria, the composition of the present invention may contain either or both of an oligosaccharide and a milk fermentation component.
In the present invention, the milk fermentation component refers to a processed product obtained by fermenting animal milk by the action of microorganisms or enzymes. In the present invention, animal milk includes milk, buffalo milk, goat milk, sheep milk, horse milk, and the like. Among them, cow's milk (milk) is economically advantageous because a large amount of raw material milk can be easily obtained. The fermented milk component can be prepared not only from milk collected from a living body but also from fractions and processed products thereof. As milk fractions or processed products, partially skim milk, skim milk, reduced whole milk, reduced skim milk, reduced partially skim milk, whey, casein, skim milk powder, whey protein concentrate (WPC), whey protein isolate (WPI) ), Butter, buttermilk, cream and the like. These processed products derived from milk are sometimes called raw milk. The raw milk can be used alone or as a raw material for milk fermentation ingredients by mixing different raw milk.

In the present invention, the milk fermentation component can be obtained as a culture obtained by fermenting milk with a microorganism. The fraction of the microorganism culture can be used as a milk fermentation component in the present invention as long as it provides a preventive effect against influenza infection when combined with propionic acid bacteria. Microorganisms added to milk for the purpose of fermentation are generally called starters. The microorganism used for the fermentation of milk is preferably lactic acid bacteria or bifidobacteria. Specifically, for example, a milk fermentation component can be obtained using lactic acid bacteria or bifidobacteria belonging to the following genera as a starter.
Genus Lactobacillus,
Streptococcus genus,
Genus Lactococcus,
More specifically, the following milk fermentation components by microorganisms are known, such as the genus Leuconostoc and the genus Pediococcus. Milk fermentation components obtained by these microorganisms are preferred as milk fermentation components in the present invention.
Lactic acid bacteria: Streptococcus lactis,
Streptococcus cremoris,
Streptococcus diacetylactis,
Enterococcus faecium,
Enterococcus faecalis,
Lactobacillus acidophilus,
Lactobacillus brevis,
Lactobacillus casei,
Lactobacillus helveticus,
Lactobacillus delbrueckii subsp.bulgaricus,
Lactobacillus delbrueckii subsp. Lactis,
Lactobacillus gasseri,
Lactobacillus mucosae,
Lactobacillus murinus,
Lactobacillus plantarum,
Lactobacillus oris,
Lactobacillus reuteri, and Lactobacillus rhamnosus
Bifidobacterium: Bifidobacterium longum,
Bifidobacterium bifidum, and Bifidobacterium breve

A method for isolating these microorganisms from nature and fermented milk is known. Alternatively, already isolated microorganisms can be obtained by distribution from a cell bank or the like. Furthermore, lactic acid bacteria starters for obtaining milk fermentation components are commercially available. Milk fermentation ingredients produced by a commercially available lactic acid bacteria starter can also be used in the composition of the present invention. Several products are sold depending on the pH and physical properties of the fermented milk produced. The physical properties of fermented milk refer to hardness and smoothness. A commercially available lactic acid bacterium starter can be used as a lactic acid bacterium starter for obtaining a milk fermentation component in the present invention as long as it promotes the induction of neutralizing antibodies by an influenza vaccine when administered with a culture of propionic acid bacteria.

In order to obtain the milk fermentation component in the present invention, the following microorganisms can be inoculated into the raw milk as a lactic acid bacteria starter.
Lactobacillus bulgaricus (L.bulgaricus),
Streptococcus thermophilus (S. thermophilus),
Lactobacillus lactis
In general production of fermented milk, raw milk may be added with one or more selected from lactic acid bacteria other than these lactic acid bacteria and yeast. However, in the present invention, in any case, a mixed starter of Lactobacillus bulgaricus (L. bulgaricus) and Streptococcus thermophilus (S. thermophilus) standardized as a yogurt starter in the Codex standard is used. preferable. When additional microorganisms are added, additional microorganisms can be mixed into the mixed starter in consideration of the fermentation temperature and fermentation conditions of the target fermented milk. Other microorganisms such as Lactobacillus gasseri (L. gasseri) and Bifidobacterium can be used as the microorganism to be additionally mixed in the mixed starter.

In the present invention, the microorganism added to the raw milk as a mixed starter can be selected from microorganisms deposited in the cell bank. Examples of desirable strains that can be used in the mixed starter are shown below.
Lactic acid bacteria starter consisting of a mixed culture of the following microorganisms:
Lactobacillus bulgaricus (L.bulgaricus JCM 1002T)
Streptococcus thermophilus (S. thermophilus ATCC 19258)
Lactic acid bacteria starter consisting of a mixed culture of the following microorganisms:
Streptococcus thermophilus OLS 3059 (FERM BP-10740)
Streptococcus thermophilus OLS3294 (NITE P-77)
Lactobacillus delbrueckii subspecies bulgaricus OLL 1073R-1 (FERM BP-10741)
Lactobacillus delbrueckii subspecies bulgaricus OLL 1255 (NITE BP-76)

Cheese, natural cheese, yogurt, fermented milk, whey fermented product, whey cheese and the like that can be obtained by fermentation of these microorganisms are included in the milk fermentation components in the present invention. Examples of the milk fermentation component for blending into the composition of the present invention include, for example, fermented milk (yogurt) with reduced water (whey) (for example, Japanese Patent No. 3,179,555). The protein derived from fermented milk (yogurt) has an amino acid score of 100, the digestibility of the protein is enhanced by fermentation, and the nutritional value is high.

Among these milk fermentation components, for example, cheese is a preferable milk fermentation component in the present invention. For example, liquid milk prepared by combining one or more liquid milk raw materials can be made curd by adding enzymes or acids after fermentation with lactic acid bacteria. Cheese is obtained by removing whey from curd. Regardless of solidification or aging, cheese is obtained by removing whey from curd. Cheese is roughly classified into ripened cheese that has undergone a ripening process after production and non-ripened cheese. In the ripening process of cheese, the propagation (fermentation) of lactic acid bacteria and mold usually proceeds, and a characteristic flavor is formed in each cheese. In contrast to ripened cheese, cheese that maintains a state in which whey is removed from the curd is unripened cheese. Non-aged cheese (fresh cheese) is preferred as a milk fermentation component in the present invention. There are many types of non-aged cheese (fresh cheese) such as cottage, quark, string, Neuchâtel, cream cheese, mozzarella, ricotta and mascarpone. Among these fresh cheeses, quark is preferably used in the present invention. The production method of quark is publicly known (for example, JP 6-228013, Cheeseheand Fermented Milk Foods, dsVolume I: Origins and Principles, Frank V. Kosikowski and Vikram V. Mistry, FV Kosikowski, LLC-161, pages 147 It is. Quark is a kind of cheese (generic name) and its nutritional composition has already been clarified (Milk and Dairy Product Technology, Edgar Spreer; Axel Mixa, Marcel Dekker Inc, 1998 pages 245-249) .

A general method for producing non-aged cheese will be described below. First, curd is produced from raw milk. After inoculating a raw milk with a starter and culturing, rennet is further added to form curd (curd). Prior to the production of the curd, the raw milk can be pretreated if necessary. For example, in order to reduce the quality difference between production lots, the quality can be adjusted by mixing many kinds of milk raw materials. Such processing is called standardization. Furthermore, a homogenize treatment that mechanically breaks fat globules in milk can be added. Alternatively, centrifugal sterilization or heat treatment can be performed to remove microorganisms mixed in the milk raw material.

The solid content obtained by separating whey from the obtained curd (curd) is non-aged cheese (fresh cheese). Methods are known for separating whey from curd by centrifugation or membrane separation. For example, a centrifuge such as a quark separator is used for separating whey. Alternatively, the separation process can be made more efficient by cutting the curd in advance or heating as necessary.

More specifically, fresh cheese that can be obtained by the following raw materials and processes is preferred as a milk fermentation component in the present invention. During the following steps, mainly Lactobacillus bulgaricus and / or Streptococcus thermophilus can be used for fermentation.
Heat pasteurize bovine skim milk;
Inoculate 0.5-5% lactic acid bacteria starter to start fermentation;
separating whey from curd formed when pH reaches 4.6;
Cooling the curd from which the whey has been separated to obtain non-aged cheese The non-aged cheese that can be produced in this way is generally sometimes referred to as Quark. An example of the composition of non-aged cheese is as follows.
17-19% total solids,
11-13% protein,
1% or less fat (ie 0-1%),
2-8% carbohydrates,
Lactose 2% or less (ie 0-2%)

The blending amount of the milk fermentation component is, for example, about 0.01 to 30%, usually about 0.1 to 20%, preferably about 0.5 to 10% of the whole composition as a protein derived from the milk fermentation component. It can mix | blend in a ratio. Alternatively, 0.01 to 33 g, 0.1 to 22 g, 0.5 to 11 g, preferably 2 to 6 g, more preferably 2.5 to 4.5 g can be blended per 100 ml of the composition. Alternatively, the milk-fermented component-derived protein contained in the entire composition of the present invention is about 0.1 to 100%, preferably about 1 to 100%, more preferably about 30% to the total protein amount of the composition. 100%.

In addition, lactic acid bacteria belonging to the genus Lactococcus can be added to heat-sterilized skim milk as a lactic acid bacteria starter, and a fermented milk fermentation component can be used as the non-aged cheese of the present invention. More specifically, a lactic acid bacteria starter mixed with the following microorganisms can also be used.
Lactococcus lactis,
Lactococcus cremoris and microorganisms belonging to the genus Leuconostoc A lactic acid bacteria starter mixed with the above microorganisms can be added to heat-sterilized skim milk and cultured to obtain curd curd. It is also possible to remove whey from the fermented milk component to make a non-aged cheese. Non-aged cheese obtained by cutting a card obtained by fermentation in advance with a cutter and separating whey while heating is also included in the milk fermentation component of the present invention.
In addition, rennet (Rennet) added to raw milk and solidified is also included in the non-aged cheese in the present invention. Rennet is a raw material for producing cheese and the like whose main component is chymosin (EC3.4.23.4).

The composition of the present invention can be administered enterally. Enteral administration refers to delivering the composition of the present invention to the intestinal tract. Therefore, not only oral administration but also administration methods using enema administration and various tube feeding are included in enteral administration. Tube feeding is a method in which a liquid food or the like is administered directly to the digestive tract through a tube to a patient who is difficult to take a meal orally. There are the following administration routes depending on the method of tube installation.
Nasal feeding
External fistula
Gastrostomy (percutaneous endoscopic gastrostomy; PEG)
Jejunostomy
External fistula refers to the passage through the digestive tract from outside the abdominal wall and includes gastrostomy and jejunostomy. Further, enema administration in which the composition is injected into the rectum from the anus is also included in the enteral administration. In the case of oral administration, the dosage form of the composition is arbitrary. However, for tube administration or enema administration, the composition of the present invention is advantageously in the form of a paste, semi-solid or liquid.

The composition of the present invention is useful for preventing influenza virus infection in animals. In the present invention, animals refer to mammals and birds that are hosts for influenza viruses. Infections of influenza viruses are known for mammals, for humans, for pigs, and the like. In addition to pigs, it can be applied to cattle, goats, sheep, horses, buffalos, camels, etc., as well as animals and livestock raised in pets and zoos. Birds are also infected with influenza viruses in poultry such as chickens and many wild birds. Any of these animal species can be expected to have an effect of preventing influenza infection by administration of the composition of the present invention. When administering the composition of this invention to an animal, the composition of this invention can be mix | blended and administered to feed.

When a milk fermented ingredient is blended with the composition of the present invention, the dosage (intake) of the milk fermented ingredient is about 1 mg to about 20 g, preferably about 10 mg to about 15 g, more preferably about 10 mg to about 15 g in solid content per kg body weight per day. Can be from about 50 mg to about 10 g. The dosage can be adjusted according to the dosage form, symptoms, sex, age, weight, and the like.
For example, when a milk fermentation component is mixed with the composition of the present invention and administered to a human, the dosage is generally about 0.05 g to about 1500 g, preferably about 0.05 g to about 1.5 g in solid content per day. About 1000 g, more preferably about 2.5 g to about 800 g. For those who need to promote the induction of neutralizing antibodies by the composition of the present invention, it can be administered at once or in divided portions and appropriately administered before meals, after meals, between meals and / or before going to bed. The dose can be appropriately adjusted individually according to the age, body weight, and purpose of administration. Further, the composition of the present invention can be used in place of a meal, and can also be used as a dietary aid.

The culture of propionic acid bacteria constituting the composition of the present invention, or milk fermentation components and oligosaccharides that can be additionally added are already used as nutrients and liquid food components. Therefore, the composition of this invention can also be mix | blended with the nutrient and liquid food containing these components. For example, a culture of propionic acid bacteria containing BGS is called “profec” and is permitted as an ingredient involved in food for specified health use. The trade names “B.G.S. powder” and “tummy vitality tablet” include “Profec” which is a fermented whey fermented with propionic acid bacteria. The trade name “Fibren YH” (manufactured by Meiji Dairies) is a liquid food containing fermented milk ingredients. Therefore, the composition of this invention can also be obtained by mix | blending these goods according to the above compounding ratios.

Furthermore, a kit for preventing influenza infection can be constituted by combining a composition containing a culture of propionic acid bacteria and a composition containing a milk fermentation component. That is, the present invention
The present invention relates to a kit for preventing influenza infection, comprising: (a) a composition comprising a culture of propionic acid bacteria; and (b) a composition comprising a milk fermentation component. Alternatively, the present invention provides a kit for promoting the induction of influenza virus neutralizing antibodies in an influenza vaccinated animal comprising the above compositions (a) and (b). The kit of the present invention can be constituted by combining, for example, a nutritional composition containing a culture of propionic acid bacteria and a nutritional composition containing a milk fermentation component. These nutritional compositions are distributed as liquid foods and nutrients.

The present inventors have found that induction of neutralizing antibodies by influenza vaccination is enhanced in humans who have ingested propionic acid bacteria cultures. That is, the present invention provides a composition comprising a culture of propionic acid bacteria, which is used for enteral administration to an animal vaccinated with an influenza vaccine. Or this invention provides the induction | guidance | derivation promoter of the neutralizing antibody of the influenza virus in the animal inoculated with the influenza vaccine containing the culture of propionic acid bacteria. In a preferred embodiment, the composition of the present invention, or the neutralizing antibody induction promoter, can additionally contain either or both of a milk fermentation component and an oligosaccharide.

In addition, the present invention relates to the use of a culture of propionic acid bacteria in the production of a promoter for the induction of neutralizing antibodies for influenza virus in animals vaccinated with influenza virus. Alternatively, the present invention relates to the use of a culture of propionic acid bacteria in promoting the induction of neutralizing antibodies in animals vaccinated with influenza. In addition, the present invention relates to a method for producing a neutralizing antibody induction promoter in an animal vaccinated with an influenza vaccine, which comprises the step of combining a culture of propionic acid bacteria and a pharmaceutically acceptable carrier.
Furthermore, the present invention relates to the use of a culture of propionic acid bacteria in the production of a preventive agent for influenza infection. Alternatively, the present invention relates to the use of propionic acid bacteria cultures in the prevention of influenza infection.

Alternatively, the present invention provides a pharmaceutical composition for promoting the induction of influenza virus neutralizing antibodies in an animal vaccinated with an influenza virus vaccine, including a culture of propionic acid bacteria. The invention further relates to the use of a culture of propionic acid bacteria in the manufacture of a pharmaceutical composition for promoting induction of influenza virus neutralizing antibodies in animals vaccinated with influenza virus. The pharmaceutical composition of the present invention comprises a pharmaceutically effective amount of a culture of propionic acid bacteria. The pharmaceutical composition of the present invention can be blended with a carrier suitable for oral administration or enteral administration. The pharmaceutical composition of the present invention can be administered also as a liquid food for the purpose of inducing neutralizing antibodies in animals vaccinated with influenza vaccine.

In the present invention, the influenza vaccine includes a vaccine that is administered for the purpose of preventing influenza virus infection and severe infection after infection. Inactivated vaccines currently in practical use in Japan and nasal vaccines in practical use overseas are included in the influenza vaccine of the present invention. Inactivated vaccines are generally administered subcutaneously, intradermally or intramuscularly. On the other hand, nasal vaccine is a live virus vaccine. The purpose of the nasal vaccine is to induce an IgA antibody that is highly effective in preventing viral infection in the airway mucosa by spraying into the nasal cavity.

Alternatively, the present invention
(1) administering a culture of propionic acid bacteria to an animal; and
(2) inoculating animals with influenza vaccine;
A method for preventing influenza infection, including
In the present invention, prevention of influenza infection specifically includes either or both prevention of influenza virus infection and prevention of severity after influenza virus infection. An influenza infection is an infectious disease with various symptoms caused by infection with an influenza virus, which is a pathogen. In the present invention, an influenza infection may be simply referred to as “influenza”. Severity of influenza infection includes the following conditions:
The infectious symptoms become serious,
Increased types of infectious symptoms Increased virus-infected tissues or cells Increased virus growth in vivo

Therefore, inhibiting at least one of these conditions means prevention of influenza infection in the present invention. In the present invention, prevention of influenza infection includes prevention of influenza virus infection. More specifically, enhancing the induction of virus neutralizing antibodies is included in the prevention of influenza virus infection. The production level of neutralizing antibodies induced in vivo by inoculation with influenza vaccine may decrease over time. Preventing the reduction of neutralizing antibodies contributes to the prevention of influenza infection. Therefore, preventing the decrease in the production level of neutralizing antibodies is also included in the promotion of induction of virus neutralizing antibodies.

This is because neutralizing antibodies contribute to the prevention of the spread of infected tissues or cells in the body of influenza virus. The animal species that can be expected to prevent influenza infection by the composition of the present invention or the method of the present invention is an influenza virus host animal. More specifically, for example, humans, animals including humans, or animals other than humans.
Moreover, the preventive effect of influenza infection caused by the composition of the present invention does not depend on the antigenicity of the influenza virus. Therefore, it is effective in preventing infection with all kinds of influenza viruses. In particular, influenza viruses belonging to type A and its subtypes are preferred as influenza viruses whose infection should be prevented in the present invention. More specifically, an influenza A virus whose host is human, swine, avian or the like is suitable as a prevention target in the present invention.

The promotion of neutralizing antibody induction can be confirmed, for example, as follows. That is, it is possible to administer the same influenza vaccine to a group to which the composition of the present invention is administered and a group to which the composition is not administered, and to compare the induction state of virus neutralizing antibodies in both groups. At this time, the members of each group are allocated so that the conditions other than the administration of the composition are equal. In other words, it is desirable to distribute conditions such as health status, age, physique, and sex ratio so that there is no bias among the groups. Ideally, genetic features should be as homogeneous as possible. Therefore, when the subject is a human, it is desirable that the race is the same group. When confirming the preventive effect in non-human animals, the genetically identical population should be used as much as possible.

Under such conditions, both populations are administered the composition and vaccinated on the same schedule. And in the group which administered the composition of this invention, when the induction | guidance | derivation of the neutralizing antibody is significantly enhanced, the influenza infection disease prevention effect of the said composition can be confirmed. Here, enhancement of neutralizing antibody induction means, for example, that one of the following can be confirmed. Among the indicators shown below, the neutralizing antibody titer can be evaluated by, for example, comparing the proportion (expression rate) of individuals exceeding the infection-preventing antibody titer between populations.
If the antibody titer of neutralizing antibody rises quickly,
When neutralizing antibody production continues for a long period of time or when a high neutralizing antibody titer is achieved Therefore, the number of individuals in which any of these effects was confirmed by administration of the composition of the present invention If it increases significantly, the preventive effect of the composition on influenza infection can be confirmed. Methods for quantitatively evaluating the neutralizing antibody titer are known. For example, the infection inhibitory effect of an antibody can be confirmed using infectious influenza virus and cultured cells. By serially diluting the antibody, the infection inhibitory action can be quantitatively compared.

In the present invention, the culture of propionic acid bacteria is administered before or after vaccination or simultaneously with vaccination. Preferably, the culture of propionic acid bacteria is administered continuously from before to after vaccination. That is, this invention provides the composition containing the culture of propionic acid bacteria used so that the animal inoculated with influenza vaccine may be continuously enterally administered before and after vaccination. Before and after vaccination means that the day of vaccination is 0 (zero) day, for example, −150 to +150 days, usually −60 to +60 days, or −8 weeks to +8 weeks.
During this period, the compositions of the invention are administered at least once per day in a pharmaceutically effective amount. The daily dose can be divided into a plurality of doses. Alternatively, it can be administered every other day. Furthermore, in this invention, the effective amount of the culture of propionic acid bacteria can also be administered in combination of multiple types of compositions having different blending ratios.

In fact, the present inventors have confirmed that the induction of neutralizing antibodies by the vaccine is promoted in humans who have taken the culture of propionic acid bacteria before and after the administration of the influenza vaccine. Therefore, the present invention provides a method for promoting or enhancing the induction of neutralizing antibodies in animals administered with influenza vaccine, comprising the following steps (1) and (2).
(1) administering a culture of propionic acid bacteria to an animal; and
(2) inoculating animals with influenza vaccine;

The composition of the present invention can be expected not only to promote the induction of neutralizing antibodies at the time of vaccination but also to enhance the induction of neutralizing antibodies during virus infection. That is, the composition of the present invention can relieve symptoms even in patients infected with influenza virus by enhancing the ability of the patient to produce virus neutralizing antibodies and suppressing the spread of virus infection in the patient. . In particular, at the time of infection with a virulent virus, the risk of causing systemic symptoms as well as respiratory tract infections is predicted. The composition of the present invention can be expected to have an effect of increasing the ability of inducing neutralizing antibodies against infectious viruses and preventing its seriousness by continuous ingestion by patients.

The composition of the present invention can be used in the form of any medicine or food and drink. For example, induction of neutralizing antibodies of influenza vaccine can be enhanced by direct administration as a pharmaceutical. Alternatively, in anticipation of the enhancement of the preventive effect of the influenza vaccine, it can be ingested as a special-purpose food such as a food for specified health use or a nutritionally functional food. Furthermore, it can be ingested as a food by adding it to various foods and drinks, regardless of the form of liquid, paste, solid, powder or the like. Foods and drinks include milk, soft drinks, fermented milk, yogurt, cheese, bread, biscuits, crackers, pizza crusts, prepared milk powder, liquid foods, food for the sick, nutritional foods, frozen foods, food compositions, processed foods, etc. Examples of such commercial foods can be given. When the composition of the present invention is in the form of an acidic pharmaceutical or food or drink, the pH can be adjusted to pH 2.0 to pH 6.0, preferably pH 3.0 to pH 5.0.

When the composition of the present invention is continuously administered to animals, it can also be administered as a nutrient, food or drink, or food. When the composition of the present invention is administered as a nutrient, food or drink, or diet, in addition to the milk fermentation component and the propionic acid bacteria culture, the nutritional composition of the composition is added by adding additional nutrients. Can be adjusted. As the additional nutrient in the present invention, water, protein, carbohydrate, lipid, vitamins, minerals, organic acid, short chain fatty acid, organic base, fruit juice, flavors and the like can be used. For these nutrients, for example, the following components can be used.

Protein: (Animal protein, plant protein, or their degradation products)
Whole milk powder, skim milk powder, partially skimmed milk powder, casein, whey, whey powder, whey protein, whey protein concentrate, whey protein isolate, α-casein, β-casein, κ-casein, β-lactoglobulin, lactoferrin, Soy protein, chicken egg protein, meat protein and other milk-derived lipids and saccharides: Butter, whey minerals, cream, non-protein nitrogen, various milk-derived components such as sialic acid, phospholipids and lactose Peptides and amino acids: Casein Peptides such as phosphopeptides, arginine, lysine, and various amino acids

Sugar: Processed starch (dextrin (maltodextrin, resistant digestive dextrin, etc.), soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), dietary fiber and other fats and oils: lard, fish oil, etc., these fractionated oils, hydrogen Animal fats and oils such as additive oils and transesterified oils; Palm oils, safflower oils, corn oils, rapeseed oils, palm oils, fractionated oils thereof, hydrogenated oils, vegetable oils such as transesterified oils, etc.

Vitamins: Vitamin A, carotene, vitamin B group, vitamin C, vitamin D group, vitamin E, vitamin K group, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, choline, folic acid and other minerals Kinds: Calcium, phosphorus, potassium, chlorine, magnesium, sodium, copper, iron, manganese, zinc, selenium, chromium, molybdenum, etc. Organic acids: Malic acid, citric acid, lactic acid, tartaric acid and other short chain fatty acids: acetic acid, propionic acid These additional nutrients such as butyric acid, valeric acid, caproic acid, etc. can be used either chemically synthesized or natural-derived components. Or the foodstuff containing the target component can also be mix | blended as a raw material. These components can be blended in combination of at least one or two or more according to the composition of the target nutrient. The form of the composition may be solid or liquid. It can also be in the form of a gel or semi-solid. Therefore, a nutrient can also be administered as a liquid food.

In fact, as shown in the examples described below, the composition of the present invention induces influenza virus neutralizing antibodies in a host animal vaccinated with influenza vaccine by ingesting it as a liquid food containing a culture of propionic acid bacteria. Promoted. Therefore, the composition which has a composition as a liquid food which mix | blended the culture of propionic acid bacteria is one of the preferable aspects in this invention. That is, this invention relates to the manufacturing method of the liquid food which promotes the induction | guidance | derivation of the neutralizing antibody of the influenza virus in the subject who ingested the influenza vaccine including the process of mix | blending the culture of propionic acid bacteria with a liquid food. Alternatively, the present invention provides a method for imparting the ability to promote the induction of neutralizing antibodies against influenza virus in a subject who has taken an influenza vaccine to the liquid diet, comprising the step of blending a culture of propionic acid bacteria with the liquid diet. Furthermore, this invention provides the prevention composition of influenza infection containing the following nutrients.
A culture of propionic acid bacteria;
Milk fermentation ingredients;
oligosaccharide;
protein;
Carbohydrates;
Lipids; and dietary fiber.

In the above composition, the culture of propionic acid bacteria is, for example, (i) 1,4-dihydroxy-2-naphthoic acid (DHNA), and (ii) 2-amino-3-carboxy-1,4-naphthoquinone (ACNQ) Can be either or both. Moreover, when the milk fermentation component in the said composition contains a protein, the protein from which it originates differently as a protein can also be further mix | blended. For example, a milk component can be blended in addition to the milk fermentation component. Similarly, saccharides other than oligosaccharides can be blended as the saccharide in the above composition.
Each component which comprises the composition of this invention can be suitably adjusted according to various conditions, such as a physique, age, sex, etc. of the object administered as a liquid food. More specifically, the following composition (per 100 mL) can be shown as a general composition.

Propionic acid bacteria culture: 1 mg to 22 g, usually 10 mg to 17 g, preferably 10 mg to 11 g; or: 0.01 μg to 15 mg in DHNA amount,
Usually 0.5 μg to 10 mg,
Preferably 0.5 μg to 0.1 mg;
Milk fermentation components: 0.01 g to 33 g, usually 0.1 g to 22 g, preferably 0.5 g to 11 g (as protein amount);
Oligosaccharide: 1 mg to 20 g, usually 50 mg to 11 g, preferably 0.1 g to 6 g;
Protein: 0.01 to 50 g, usually 0.1 to 30 g, preferably 0.5 to 15 g;
Carbohydrate: 0.1 g to 40 g, usually 0.5 g to 30 g, preferably 1 g to 25 g;
Lipid: 0.1 to 20 g, usually 0.3 to 15 g, preferably 0.6 to 10 g; and dietary fiber: 0 to 15 g, usually 0 to 10 g, preferably 0 to 8 g.
In the above composition, when a culture of propionic acid bacteria containing DHNA is blended, the amount of the culture can be blended so as to have the above composition in terms of DHNA. Similarly, the oligosaccharide blended in the above composition can be blended as part of the carbohydrate. That is, the oligosaccharide constitutes a part of the carbohydrate in the above composition.

In addition, the composition of the present invention may further contain at least one nutrient selected from the group consisting of vitamins, minerals, organic acids or short-chain fatty acids, and organic bases. The composition when these components are blended can be appropriately adjusted according to various conditions such as the physique, age, and sex of the subject to be administered as a liquid food. More specifically, the following composition (per 100 mL) can be shown as a general composition.
Vitamins: 0-2 g, usually 0-1.5 g, preferably 0-500 mg;
Minerals: 0-5g, usually 0-3g, preferably 0-2g:
Organic acid or short chain fatty acid: 0 to 5 g, usually 0 to 3 g, preferably 0 to 2 g
That is, this invention provides the manufacturing method of the liquid food for enhancing the induction | guidance | derivation of the neutralizing antibody by influenza vaccine including the process of mix | blending the said nutrient with said composition. In the liquid food produced by the present invention, the liquid food is administered to the subject to be inoculated with the influenza vaccine before and after the inoculation of the influenza vaccine, thereby promoting the induction of neutralizing antibodies against influenza virus in the subject. Can be displayed.

The composition of the present invention can be prepared by blending a propionic acid bacteria culture with, for example, a pharmaceutically acceptable carrier. Furthermore, when adding an additional component, the above-mentioned saccharide | sugar, protein, lipid, etc. are mixed as a carrier or a liquid food, and it mixes and prepares. Usually, the milk-fermented component and the protein derived from the propionic acid bacterium culture are preferably about 1% by weight or more of the protein in the composition, for example, about 30% by weight or more, preferably It can be prepared so that it may become the ratio of 70 weight% or more of the protein in a composition, More preferably, it may become a ratio of about 100 weight%. When the composition of the present invention is prepared as a liquid food, it is desirable to adjust it to 0.1 to 3 kcal per ml, preferably 0.7 to 2 kcal. Moreover, at the time of mixing, at least 1 or more of the additional components which consist of vitamins, minerals, and dietary fiber can also be added. Dietary fiber is divided into water-soluble dietary fiber and insoluble dietary fiber, and both can be used. Specifically, examples of the water-soluble dietary fiber include the following components.
Pectin (protopectin, pectinic acid, pectinic acid),
Guar gum hydrolyzate,
Glucomannan,
Galactomannan,
Psyllium,
Corn fiber,
Alginic acid,
Alginate degradation product,
Caraginan,
Indigestible dextrin Examples of insoluble dietary fiber include crystalline cellulose, beet fiber, wheat bran and the like. Preferably, pectin, guar gum hydrolyzate, and indigestible dextrin can be used. Furthermore, depending on the case, a fragrance | flavor and another compound can also be added.

After mixing the various components as described above, the container is filled and subjected to heat sterilization as necessary to obtain a liquid food or enteral nutrient. When the pH of the composition is acidic, the heat treatment can be performed under milder conditions than usual. For example, the heat sterilization treatment of a neutral liquid food is a retort sterilization condition of 120-130 ° C., 20-40 minutes, an indirect sterilization condition of 140-145 ° C., 4-10 seconds. -30 minutes of retort sterilization or indirect sterilization at 95-110 ° C. for 20-60 seconds. By mild sterilization conditions, the flavor is improved and the addition of heat-sensitive ingredients is allowed. In addition, the composition of the present invention can be made into a gel form with agar or gelatin, can be made into a granular food / medicine by spray drying or the like, or can be made into a solid food / medicine.

The composition of the present invention can be produced by a known method in the fields of liquid foods and enteral nutrients. For example, after sterilizing the liquid composition in advance and then aseptically filling the container (for example, a method using a combination of the UHT sterilization method and the aseptic packaging method), or after filling the container with the liquid composition A method of sterilization by heating with a container (for example, a retort method, an autoclave method) or the like can be employed. That is, when the composition is used in a liquid form, a homogenized product based on the composition (a homogenized sterilizing solution) is again heat-sterilized at about 120 to 145 ° C. for about 1 to 10 seconds as necessary. Then, cool and aseptically fill, or fill into cans and soft bags and sterilize by retort. And when the usage form of a composition is a powder, the said homogenized material is spray-dried or freeze-dried, for example.

Hereinafter, the present invention will be described in detail, but the present invention is not limited to the individual forms described below. In the present invention, when the raw materials are prepared (added / mixed), they are heated and prepared. This is because if the preparation temperature is high, such as 90 ° C., the protein will coagulate (curd), and if the preparation temperature is low, such as 2 ° C., the protein will be difficult to dissolve or disperse in water. Therefore, as a preparation step, the temperature is preferably 5 to 85 ° C., more preferably 15 to 75 ° C., further preferably 25 to 55 ° C., and particularly preferably 35 to 50 ° C. At this time, it is preferable to adopt an appropriate preparation time while taking into consideration the growth of bacteria (contaminating bacteria, etc.) in the preparation liquid.

Furthermore, in the present invention, the mixture is sterilized at high temperature and then homogenized. This is because high-temperature sterilization (heating) may denature proteins and increase viscosity (thickening), but homogenization after high-temperature sterilization can reduce the degree of thickening. Here, homogenization after high-temperature sterilization means homogenization after high-temperature sterilization and before filling into a container or the like to make a product, and the number of times is not limited to one, but may be two or more. There may be. For example, if the preparation liquid is sterilized and then sterilized for the second time, it is homogenized after the second sterilization. Further, when the preparation liquid is sterilized after being sterilized and further sterilized for the second time, it is homogenized again for the second time after sterilization for the second time. And it will homogenize after sterilizing a preparation liquid, and may homogenize in the second time anew without sterilizing. That is, in the present invention, after pasteurizing the preparation liquid at high temperature, it is important to homogenize even once before filling into a container or the like to make a product.

On the other hand, even after the high-temperature sterilized preparation liquid (sterilizing liquid) is homogenized, it may be sterilized again as long as the sterilizing liquid does not thicken. For example, the preparation liquid may be sterilized after being sterilized and then sterilized a second time without being sterilized at high temperature. At this time, as the high temperature sterilization step, for example, the temperature is 100 to 150 ° C., the holding time is 1 to 30 seconds, preferably 115 to 145 ° C., 1 to 20 seconds, more preferably 120 to 145 ° C., 1 to 10 seconds, A thermal history corresponding to 125 to 140 ° C. and 1 to 5 seconds is more preferable. When sterilized at high temperature, the protein is denatured and the sterilizing solution tends to thicken. In other words, the liquid mixture after sterilization is hard to thicken unless sterilized at high temperature. Therefore, the effect of reducing the degree of thickening by homogenization can be said to be particularly effective when performing high-temperature sterilization.

Moreover, when sterilizing at high temperature, the pressure may be adjusted (pressurized or depressurized) to the preparation liquid. At this time, for example, the sterilization pressure is usually about 1 to 10 kg / cm ² for the purpose of preventing boiling of the preparation liquid. That is, in the high temperature sterilization of the present invention, such pressure may be applied in addition to temperature (heating). Examples of the high-temperature sterilizer include a plate heat exchanger, a tube heat exchanger, a steam injection sterilizer, a steam infusion sterilizer, and an electric heating sterilizer. On the other hand, when homogenizing, using a homogenizer, for example, when the temperature is set to about 10 to 60 ° C. and the flow rate is set to about 100 to 10000 L / h, the pressure is set to 10 to 100 MPa, preferably 20 to 80 MPa, more preferably. Is 30 to 70 MPa, more preferably 20 to 50 MPa. Further, if necessary, the treatment conditions may be changed multiple times by changing the operation conditions such as high temperature sterilization and homogenization.

Hereinafter, the present invention will be described with further detailed examples, but the present invention is not limited thereto. In the blending process, warm water at the above temperature is stirred in the tank, and the ingredients other than vitamin mix (mixed ingredients of vitamins) are added, mixed and stirred sequentially in consideration of ease of mixing and diffusing. To prepare a mixed solution. The order in which the raw materials are easily mixed and diffused depends on the amount and characteristics of the raw materials. Therefore, depending on the composition, various blending components can be added in various orders at once or divided. Specifically, for example, there is a method in which sugar, protein, fat and oil are added in this order. Another example is a method in which some sugars, proteins, other sugars, minerals, and fats are added in this order. As another example, there is a method in which oils and fats, proteins, sugars, and minerals are added in this order. This prepared solution was sterilized by heating with a steam injection method, and then homogenized with a homogenizer (homogenized with two-stage pressure) to obtain a sterilizing solution. Vitamin mix (mixed ingredient of vitamins), flavor (fragrance) and the like were added to and mixed with this sterilizing solution to obtain a final sterilizing solution. The final sterilizing solution was further heat sterilized (two-stage sterilization) using a steam infusion method, and then homogenized (homogenized with two-stage pressure) using a homogenizer to obtain a composition.

The composition of the present invention can be used as an enteral nutrient having a preventive effect on influenza infection. That is, when the composition of the present invention is mixed with a liquid food or enteral nutrient, the liquid food or enteral nutrient itself has a preventive effect on influenza infection. Can be used for preventive purposes. As foods having at least one of the following functions, for example, it can be used as health functional foods such as foods for specified health use and functional foods for nutrition.
Preventive action against influenza infection,
Neutralizing antibody induction enhancing action, or neutralizing antibody titer lowering prevention action after vaccination In facilities with many elderly people and hospitalized patients, it is considered to be a preventive measure to increase the infection prevention effect of influenza vaccine. Alternatively, it can be used as a baby food for preventing influenza for infants and a nutritional food for preventing influenza infection for general public. Furthermore, in a desirable embodiment, the composition of the present invention can be a composition having an intestinal action. Therefore, it can also be used as a dietary supplement or a food for preventing influenza infection that also has an intestinal regulating action.
In addition, all prior art documents cited in the present specification are incorporated herein by reference. Hereinafter, the present invention will be described more specifically based on examples.

Example 1: Antibody titer measurement after influenza vaccination for tube feeding patients:
[Method]
Tube feeding patients were divided into two groups, a test group and a control group, and the influence of the culture of propionic acid bacteria on the induction of neutralizing antibodies by influenza vaccine was evaluated. Table 1 summarizes the patient background of the control group and the test group. When analyzed by Students' t test (equal variance) or Welch's test (unequal variance), there was no significant difference between groups in the patient background.

Patient background in the control and test groups

Statistical significance was analyzed by Student's test (equal variance) or Welch's test (unequal variance).

The liquid food administered to each group is as follows.
Control group (11 persons): General composition liquid food Test group (11 persons; however, analysis of fecal score and bacterial flora was conducted on 12 persons): Test liquid food and culture of propionic acid bacteria The “general composition liquid food” is a liquid food that contains milk protein as protein and does not contain a milk fermentation component or a culture of propionic acid bacteria.
On the other hand, the test liquid food is a liquid food containing a milk fermentation component (3.8 g / 100 kcal) as a protein. The fermented milk components contained in the test liquid food are sterilized by adding honey, vitamins, minerals, edible fats, dietary fiber, and dextrin to fermented and concentrated skim milk with L. bulgaricus and S. thermophilus. is there.
Furthermore, the culture of propionic acid bacteria is a culture of Propionibacterium freudenreich, which is a propionic acid bacterium. Further, the culture of propionic acid bacteria contains DHNA (1,4-dihydroxy-2-naphthoic acid), which is a component derived from the culture of propionic acid bacteria. Co-administration of the test liquid diet and the culture of propionic acid bacteria was considered as the administration of a novel nutritional composition comprising a milk fermentation component and a culture of propionic acid bacteria. Further, galactooligosaccharide was used as the oligosaccharide.

There is no essential difference between the general composition liquid food and the test liquid food except that the test liquid food contains a milk fermentation component. At the end of the examples, the composition (average) of these liquid foods was shown (Table 6). Each composition was administered so that there was no significant difference on a calorie basis with the control group (general composition liquid food administration group) and the test group (test liquid food administration group). As a result, the following amounts of the fermented milk components and propionic acid bacteria culture were administered to the patients in the test group, respectively.
Milk fermentation ingredients: average 33g / day
Propionic acid bacteria culture: 1 g / day on average (about 13 μg / day when converted to DHNA, or 1.6 μg per 100 kcal calorie intake)

The day on which administration of the above liquid food was started for each group was −4 weeks (at the time of grouping), and after 4 weeks (week 0), influenza vaccine (H1N1, H3N2, and B vaccines) (Kakekenken) 0.5 ml was inoculated subcutaneously in the upper arm (FIG. 1). Bifidobacteria in feces were counted 4 weeks before and 4 weeks after vaccination, and intestinal flora was compared. Blood was collected from all subjects in each group according to the following schedule, and the antibody titer against the vaccine and the cytokine concentration in the blood were measured. To measure blood cytokine levels, blood was drawn according to the following schedule;
The days divided into groups (-4 weeks),
At the time of influenza vaccination (week 0), and 2 weeks (2 weeks) and 6 weeks (6 weeks) after vaccination
Neutralizing antibodies were measured by the hemagglutination inhibition test (HI method), blood cytokines were measured by ELISA, and bifidobacteria counts in the stool were measured by real-time PCR. The measurement results were compared between two groups, before and after grouping, and between the second and sixth weeks of vaccination. At this time, the antibody titer less than 10 was set to 5.
Further, the fecal score value was recorded daily according to the Bristol stool shape scale (Table 2) from the time of grouping (-4 weeks), and analyzed by the average score for each patient for one week. The Bristol stool shape scale has seven stages from 1 to 7, with 1 being the hardest stool, 7 being watery stool, and 3 and 4 being normal stools.

Statistical analysis was performed as follows. The statistically significant difference in the expression rate of anti-infection antibody was analyzed by chi-square test. Regarding the neutralizing antibody titer, after calculating log10 (antibody titer), the statistically significant difference between the two groups was analyzed by Mann-Whitney U test, and the analysis after vaccination was analyzed by Wilcoxon signed rank sum test. For intestinal flora and blood cytokine levels, statistically significant differences between the two groups were analyzed using the Student's test (equal variance) or Welch's test (non-equal variance). Statistical significance of each week was analyzed by paired t test. Statistically significant difference in fecal score was analyzed by Mann-Whitney 検定 U test between two groups, and statistically significant difference before and during the test was analyzed by Wilcoxon signed rank sum test.

[Table 2] Bristol Stool Chart; Bristol Stool Chart
================================================== ==========
Type 1: Separate hard lumps, like nuts (hard to pass).
Type 2: Sausage-shaped but lumpy.
Type 3: Like a sausage but with cracks on its surface.
Type 4: Like a sausage or snake, smooth and soft
Type 5: Soft blobs with clear-cut edges (passed easily).
Type 6: Fluffy pieces with ragged edges, a mushy stool.
Type 7: Watery, no solid pieces. Entirely Liquid.
================================================== ==========

[result]
The anti-infection antibody titer was set to 40 or more (Kojimahara N, et al., Vaccine. 2006; 24: 5966-9., Scharpe J, et al., Am J Kidney Dis. 2009; 54: 77-85.) The expression rate of the anti-infection antibody at 2 weeks and 6 weeks after vaccination was determined for subjects whose antibody titers were less than 40 at the average value at the time of division (-4 weeks) and at the time of vaccination (week 0). The obtained results were analyzed by chi-square test. Regarding the antigens of H1N1 and H3N2, the test group had a higher expression rate of the anti-antibody titer in the test group than in the control group at both the second and sixth weeks. Regarding the B-1 antigen, the expression rate was 9% in both groups at 6 weeks, but the test group had a higher expression rate than the control group at 2 weeks. Further, when a significant difference test was performed between the two groups, the infection blocking antibody titer expression rate of the test group was significantly high at the sixth week of the H3N2 antigen (Table 3).
Therefore, through this comparative experiment, it was revealed that the induction of neutralizing antibodies by influenza vaccine was enhanced by simultaneous intake of milk fermentation components and cultures of propionic acid bacteria.

Incidence of anti-infection antibody

Comparing neutralizing antibody titers after influenza vaccination between

weeks

2 and 6, antibody titers against H1N1 and H3N2 antigens were significantly lower at 6 weeks than at 2 weeks. . On the other hand, in the test group, the neutralizing antibody titer did not change between 2 weeks and 6 weeks (FIG. 2A, FIG. 2B).
Next, when analyzing the intestinal flora before and after influenza vaccination, the number of intestinal Bifidobacterium bacteria (log10 (number of bacteria / g stool)) in the test group was 6.39 ± 1.91 to 7.37 ± 2.40. However, there was no change from 6.65 ± 2.97 to 6.18 ± 2.80 in the control group (Table 4).

Change in the number of intestinal bifidobacteria (unit: logarithm of the number of bacteria per gram of fecal weight)
[Table 4]
===============================================
Control group Test group
-----------------------------------------------
Test start time 6.65 ± 2.97 6.39 ± 1.91
2 months after the start of the test 6.18 ± 2.80 7.37 ± 2.4
===============================================

In addition, the fecal score values at -1, 0, 1, 2, and 6 weeks in the test group were significantly lower than those at the time of grouping (-4 weeks), and improved fecalness was observed. . On the other hand, the score value during the administration period of the control group did not significantly decrease compared to the time of administration start (FIG. 3). Moreover, regarding the fecal score, the test group significantly decreased at the 3rd and 6th weeks compared to the control group.
Regarding changes in blood cytokine concentration, no significant change was observed between the test group and the control group for any cytokine. However, IL-7 was significantly increased at 6 weeks in the test group compared to before intake (-4 weeks). In the control group at this time, there was no change in the IL-7 concentration between the -4th week and the 6th week.
[IL-7]
In the test group, IL-7 concentration increased significantly at 6 weeks compared to -4 weeks (at the time of grouping or at the first blood collection), but in the control group, there was no significant change compared to -4 weeks. (FIG. 4). There was no significant difference between the control group and the test group.
[IL-17]
In the test group and the control group, the IL-17 concentration decreased at the sixth week compared with the fourth week (at the time of grouping or at the first blood collection) (control group: p <0.05, test group: p> 0.05). (FIG. 4). There was no significant difference between the control group and the test group.
[TGF-β1]
In both groups, the blood TGF-β1 concentration did not change significantly during the administration period compared to the fourth week. On the other hand, compared with the control group, the test group significantly increased at 0 and 2 weeks. However, at the time of grouping (-4 weeks), the test group already had a higher blood TGF-β1 concentration than the control group (p = 0.074). It was considered that there was no difference in the effects on the environment (Figure 4).

From the above results, it was clarified that the combined administration of the milk fermentation component and the propionic acid bacteria culture has a specific enhancing action on the induction of neutralizing antibodies upon influenza vaccination. Furthermore, the combined administration of milk fermentation ingredients and propionic acid bacteria cultures showed a clear intestinal regulating effect.

Example 2: Method for producing a new liquid food In the preparation process, warm water is stirred in a tank, and consideration is given to the ease of mixing and diffusing raw materials (Table 5) other than vitamin mix (vitamin mixed component) therein. Then, the fats and oils, milk fermentation component protein, sugar, mineral, and propionic acid bacteria culture were added in this order. The milk fermentation component was prepared by lactic acid fermentation using the following microorganism as a lactic acid bacteria starter.
Streptococcus thermophilus OLS 3059 (FERM BP-10740), and Lactobacillus delbrueckii subsp. Bulgaricus OLL 1073R-1 (FERM BP-10741)
This prepared solution was sterilized by heating with a steam injection method, and then homogenized with a homogenizer (homogenized with two-stage pressure) to obtain a sterilizing solution. Vitamin mix (mixed component of vitamins) and flavor (fragrance) were added to and mixed with this sterilizing solution to obtain the final sterilizing solution. The final sterilizing solution was further heat sterilized (two-stage sterilization) using a steam infusion method, and then homogenized (homogenized with two-stage pressure) using a homogenizer to obtain a composition. The quality and flavor of this composition were good.

[Table 5]
Composition of new liquid food:
(Both are compositions per 100kcal)
================================
New liquid food (average composition)
-------------------------------
Protein (g) 3.8
Lipid (g) 2.7
Carbohydrate (g) 14.6
Dietary fiber (g) 1.6
Ash content (g) 0.7
Vitamins:
Vitamin A (μg) 109.4 (*)
Vitamin B1 (mg) 0.1
Vitamin B2 (mg) 0.2
Vitamin B6 (mg) 0.3
Vitamin B12 (μg) 0.6
Vitamin C (mg) 15.4
Vitamin D (μg) 0.5
Vitamin E (mg) 2.9
Vitamin K (μg) 1.9
Niacin (mg) 1.5
Folic acid (μg) 48.0
Pantothenic acid (mg) 0.6
Minerals Sodium (mg) 96.6
Calcium (mg) 77.0
Iron (mg) 1.0
Phosphorus (mg) 81.9
Magnesium (mg) 19.2
Potassium (mg) 99.8
Copper (μg) 48.0
Zinc (mg) 1.0
Chlorine (mg) 105.6
-------------------------------
Protein derived from milk fermentation ingredients (g) 3.8
DHNA (μg) 1.6
Oligosaccharide (total g) 0.5
================================
(*): The amount of vitamin A was represented by retinol equivalent.

Example 3 Method for Producing a Novel Liquid Food A composition was obtained in the same manner as in Example 2 except that the milk fermentation component was prepared by lactic acid fermentation using the following microorganism as a lactic acid bacteria starter. Similar to Example 2, the quality and flavor of this composition were good.
Microorganisms used as lactic acid bacteria starter:
Streptococcus thermophilus OLS3294 (NITE P-77) and Lactobacillus delbrueckii subspecies bulgaricus OLL 1255 (NITE BP-76)

Example 4: Method for producing a novel liquid food The milk fermented components were mixed with lactic acid bacteria starter (Lactobacillus bulgaricus, Lactobacillus bulgaricus inoculated for the production of "Meiji Bulgaria Yogurt" manufactured by Meiji Dairies) and Streptococcus thermo A composition was obtained in the same manner as in Example 2 except that it was prepared by lactic acid fermentation using Philus (using Streptococcus thermophilus). Similar to Example 2, the quality and flavor of this composition were good.

[Table 6]
Composition of liquid food used for comparison:
(Both are compositions per 100kcal)
================================================== ======
General composition Liquid food Test liquid food (Average composition) + Propionic acid bacteria culture (Average composition)
-------------------------------------------------- -----
Protein (g) 3.9 3.8
Lipid (g) 3.0 2.7
Carbohydrate (g) 14.9 14.6
Dietary fiber (g) 1.0 1.6
Ash content (g) 0.4 0.7
Vitamins:
Vitamin A (μg) 69.9 109.4 (*)
Vitamin B1 (mg) 0.2 0.1
Vitamin B2 (mg) 0.2 0.2
Vitamin B6 (mg) 0.3 0.3
Vitamin B12 (μg) 0.5 0.6
Vitamin C (mg) 20.2 15.4
Vitamin D (μg) 0.7 0.5
Vitamin E (mg) 2.4 2.9
Vitamin K (μg) 6.0 1.9
Niacin (mg) 1.9 1.5
Folic acid (μg) 41.3 48.0
Pantothenic acid (mg) 0.9 0.6
Minerals Sodium (mg) 75.7 96.6
Calcium (mg) 56.7 77.0
Iron (mg) 0.9 1.0
Phosphorus (mg) 54.9 81.9
Magnesium (mg) 23.5 19.2
Potassium (mg) 72.2 99.8
Copper (μg) 86.4 48.0
Zinc (mg) 1.0 1.0
Chlorine (mg) 83.1 105.6
-------------------------------------------------- -----
Protein derived from milk fermentation ingredients (g)-3.8
DHNA (μg)-1.6
Oligosaccharide (total g) 0.1 0.5
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(*): The amount of vitamin A was represented by retinol equivalent.

The composition containing a culture of propionic acid bacteria provided by the present invention can be used as a preventive agent for influenza infection by administering it together with a milk fermentation component. The composition of the present invention enhances the induction of influenza virus neutralizing antibodies in influenza vaccinated animals. Therefore, if the composition of the present invention is administered to an animal to be inoculated with an influenza vaccine, the vaccine can become more serious or prevent infection. The composition of the present invention can also be formulated into a nutritional agent such as a liquid food or a food to provide a composition for oral intake that can be expected to have an influenza preventive action. The composition of the present invention may further contain a milk fermentation component and an oligosaccharide. By administering the composition, it is possible to expect an intestinal regulating action.

Claims

A composition for preventing influenza infection, comprising a culture of propionic acid bacteria.
The composition according to claim 1, wherein the propionic acid bacterium is Propionibacterium freudenreichii.
The composition according to claim 1, further comprising milk fermentation components and / or oligosaccharides.
The composition according to claim 3, wherein the milk fermentation component is milk obtained by fermenting milk with one or both of lactic acid bacteria belonging to the genus Lactobacillus and lactic acid bacteria belonging to the genus Streptococcus, or a mixture thereof.
The composition according to claim 3, wherein the milk fermentation component is non-aged cheese.
The composition according to claim 3, wherein at least one of the sugars constituting the oligosaccharide is galactose.
The composition according to claim 3, wherein the composition contains a culture of propionic acid bacteria and a milk fermentation component, both of which are sterilized.
The composition according to claim 1, which is used for enteral administration to an animal to be vaccinated with an influenza vaccine.
The composition of claim 3 comprising the following nutrients;
A culture of propionic acid bacteria;
Milk fermentation ingredients;
oligosaccharide;
protein;
Carbohydrates;
Lipids; and dietary fiber.
10. The composition of claim 9, further comprising at least one nutrient selected from the group consisting of vitamins, minerals, organic acids, and organic bases.
Induction promoter of neutralizing antibody of influenza virus in an animal vaccinated with influenza vaccine, including a culture of propionic acid bacteria.
A method for preventing influenza infection comprising the following steps;
(1) administering a propionic acid bacteria culture to an animal; and
(2) A step of inoculating an animal with an influenza vaccine.
The method for preventing influenza infection according to claim 12, wherein at least one step (1) is performed before, after or simultaneously with the step (2).
The method for preventing influenza infection according to claim 12, wherein in step (1), the culture of propionic acid bacteria is administered together with one or both of a milk fermentation component and an oligosaccharide.
A composition comprising the following components (a)-(c);
(a) an oligosaccharide,
(b) milk fermentation ingredients, and
(c) Propionic acid bacteria culture.
The composition according to claim 15, wherein at least one of the sugars constituting the oligosaccharide is galactose.
The composition according to claim 15, which is a composition for intestinal regulation.